European Genome-Phenome Archive

File Quality

File InformationEGAF00002445053

File Data

Base Coverage Distribution

This chart represents the base coverage distribution along the reference file. Y-axis represents the number of times a position in the reference file is covered. The x-axis represents the range of the values for the coverage.

Data is represented in a log scale to minimise the variability. A high peak in the beginning (low coverage) and a curve descending is expected.

821 649237 665126 62199 43277 34567 59262 12356 89550 43346 17344 54041 98840 58837 87136 53136 07634 55533 28931 60131 03230 68029 97828 61228 64327 08726 72026 86825 94425 28125 22324 56324 01923 20923 62123 32222 55723 00921 83221 20721 54120 42620 91920 39620 22520 02219 48419 67918 36818 74218 44617 69418 37817 94117 48517 31716 88816 84316 01016 05815 38614 84115 10714 55713 93914 48814 01514 10313 98113 45412 90912 83312 40612 57512 54212 45912 16111 52511 72511 35111 07610 97810 92610 59210 58210 23610 1889 9349 7849 9489 3579 4369 0379 1348 9028 5848 8158 6738 4648 1078 0008 1277 6447 6257 4797 3347 2267 0247 0146 5266 6496 3696 4426 1266 1805 7945 9315 6115 4155 3555 2825 2405 1625 2034 9074 9624 7834 5204 4274 3294 3964 1754 1483 9714 0173 9093 7613 6893 6143 7873 6343 6243 3833 3483 2823 4363 1373 1352 9003 0602 9873 1072 9422 8302 6582 7792 5822 6292 6642 6562 5082 2812 3502 3542 2832 1162 3842 1452 2462 1342 0492 1102 1821 9162 0211 9281 9391 9472 0391 8631 9971 8081 7631 7511 7601 5791 6411 6221 6001 5481 5521 4871 4841 3291 3581 4101 2641 2711 2051 2811 2601 2191 2101 1511 1061 2401 1131 2401 2081 1941 1531 0901 0211 0141 0301 0099311 0039378968668918178378458518917616928087757357917257276926427016776386015405756565765686385284985425356125395534804494294665335134734444134154524454234014583854093873654724023703793723703853613403443403523834363183603613033133054092972623033013372803013332372642142292642562422432382112592242472182472522512372523032442462261931962712742042432041911882161821871651741731702162191721681621571671671591221451371571551371741561421411301281471231301371451661421281301271001171061261151139199103126109122104999410882100941289583831309086701131008893897910489104699686901587468867710277859373729390958986929990106686982659189102658013678757981797665808778425963596248135727252433147524955555851775649425647615548566645515345433967546612239415251545760524247395531416137353643504130116514039374829334232385433353754374038384048762840273150513933364442393025374134333327314231333919353636312924293938432735322539264234252927298327312324892711203027222725413222322524411920223131302531343120342133252926614322273925271717243128261626251928222922222025272320222427302614192125241619322335232322182023281626151823112521302017223717291426252929322926142717151421121823232421261825414020216117162118182121151519202620221912222523241122162320221920211721141910162012112015231420191715171618221914514171012181320221346141319132114101918131611151315282826118131612813151511171614252191614917121517152013202385151212171417171219141111816196152022121714111912111616101313931014991717176161771779142769146101291312161171039151017811111211615141312107171215109211417151271314161416688815915227111192218211514121517251115151126921121813111611171913141181415241417191420171081091191316121414151018127121576101211171916161512101412106 004100200300400500600700800900>1000Coverage value101001k10k100k# Bases

Base Quality

The base quality distribution shows the Phred quality scores describing the probability that a nucleotide has been incorrectly assigned; e.g. an error in the sequencing. Specifically, Q=-log10(P), where Q is the Phred score and P is the probability the nucleotide is wrong. The larger the score, the more confident we are in the base call. Depending on the sequencing technology, we can expect to see different distributions, but we expect to see a distribution skewed towards larger (more confident) scores; typically around 40.

3 11200000000000009 425 5630000000804 21300008 277 2200000021 154 957000143 527 08500000510152025303540Phred quality score0M20M40M60M80M100M120M140M# Bases

Mapped Reads

Number of reads successfully mapped (singletons & both mates) to the reference genome in the sample. Genetic variation, in particular structural variants, ensure that every sequenced sample is genetically different from the reference genome it was aligned to. Small differences against the reference are accepted, but, for more significant variation, the read can fail to be placed. Therefore, it is not expected that the mapped reads rate will hit 100%, but it is supposed to be high (usually >90%). Calculations are made taking into account the proportion of mapped reads against the total number of reads (mapped/mapped+unmapped).

89.5 %2 184 99189.5 %10.5 %

Both Mates Mapped

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. This chart shows the fraction of reads in pairs where both of the mates successfully map to the reference genome. .

Notice that reads not mapped to the expected distance are also included as occurs with the proper pairs chart.

89.1 %2 177 52489.1 %10.9 %

Singletons

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. If one mate in the pair successfully maps to the reference genome, but the other is unmapped, the mapped mate is a singleton. One way in which a singleton could occur would be if the sample has a large insertion compared with the reference genome; one mate can fall in sequence flanking the insertion and will be mapped, but the other falls in the inserted sequence and so cannot map to the reference genome. There are unlikely to many such structural variants in the sample, or sequencing errors that would cause a read not to be able to map. Consequently, the singleton rate is expected to be very low (<1%).

0.3 %7 4670.3 %99.7 %

Forward Strand

Fraction of reads mapped to the forward DNA strand. The general expectation is that the DNA library preparation step will generate DNA from the forward and reverse strands in equal amounts so after mapping the reads to the reference genome, approximately 50% of them will consequently map to the forward strand. Deviations from the 50%, may be due to problems with the library preparation step.

50 %1 221 28150 %50 %

Proper Pairs

A fragment consisting of two mates is called a proper pair if both mates map to the reference genome at the expected distance according to the reference genome. In particular, if the DNA library consists of fragments ~500 base pairs in length, and 100 base pair reads are sequenced from either end, the expectation would be that the two reads map to the reference genome separated by ~300 base pairs. If the sequenced sample contains large structural variants, e.g. a large insertion, where we expect the reads mapping with a large separation would be a signal for this variant, and the reads would not be considered as proper pairs. Based on the sequencing technology, there is also an expectation of the orientation of each read in the fragment.

The rate of proper pairs is expected to be well over 90%; even if the mapping rate itself is low as a result of bacterial contamination, for example.

71 %1 734 95671 %29 %

Duplicates

PCR duplicates are two (or more) reads that originate from the same DNA fragment. When sequencing data is analyzed, it is assumed that each observation (i.e. each read) is independent; an assumption that fails in the presence of duplicate reads. Typically, algorithms look for reads that map to the same genomic coordinate, and whose mates also map to identical genomic coordinates. It is important to note that as the sequencing depth increases, more reads are sampled from the DNA library, and consequently it is increasingly likely that duplicate reads will be sampled. As a result, the true duplicate rate is not independent of the depth, and they should both be considered when looking at the duplicate rate. Additionally, as the sequencing depth in increases, it is also increasingly likely that reads will map to the same location and be marked as duplicates, even when they are not. As such, as the sequencing depth approaches and surpasses the read length, the duplicate rate starts to become less indicative of problems.

12.4 %302 70712.4 %87.6 %

Mapping Quality Distribution

The mapping quality distribution shows the Phred quality scores describing the probability that a read does not map to the location that it has been assigned to (specifically, Q=-log10(P), where Q is the Phred score and P is the probability the read is in the wrong location). So the larger the score, the higher the quality of the mapping. Some scores have a specific meaning, e.g. a score of 0 means that the read could map equally to multiple places in the reference genome. The majority of reads should be well mapped, and so we expect to see this distribution heavily skewed to a significant value (typically around 60). It is not unusual to see some scores around zero. Reads originating from repetitive elements in the genome will plausibly map to multiple locations.

389 9692 0385853 9949678332 1312 4388305 0751 5591 6575 3652 0029453 6506479201 4701 7675493 4231 6312 1643 9216 5081 13421 3991 0603 9411 8841 5161 0604 3725651 2121 4931 8058985 19249 8412 0711 7992 1824 4333 0532 4532 6752 9915 5094 6346 23123 6921 3245 4703 1721 3829 0303 5181 0081 875 716051015202530354045505560Phred quality score0.2M0.4M0.6M0.8M1M1.2M1.4M1.6M1.8M# Reads

Mapped vs Unmapped

Stacked column chart for both mapped and unmapped reads along all chromosomes in the reference file. It is a similar representation as shown in the Mapped reads chart but for each chromosome. Although sequenced sample may be a female, it is possible to get reads in the Y chromosome as there are common regions in both chromosomes called pseudoautosomal regions (PAR1, PAR2).

Unmapped reads belonging to each chromosome are determined when the one mate/pair is aligned and the other is not. The unmapped read should have chromosome and POS identical to its mate. It could also be due when aligning is performed with bwa as it concatenates all the reference sequences together, so if a read hangs off of one reference onto another, it will be given the right chromosome and position, but it also be classified as unmapped.

99.7%99.66%99.69%99.59%99.49%99.72%99.64%99.69%99.67%99.49%99.72%99.79%99.68%99.73%99.68%99.52%99.53%99.74%99.75%99.74%99.83%99.36%90.21%99.91%0.3%0.34%0.31%0.41%0.51%0.28%0.36%0.31%0.33%0.51%0.28%0.21%0.32%0.27%0.32%0.48%0.47%0.26%0.25%0.26%0.17%0.64%9.79%0.09%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped